Uncoupled IP3 receptor can function as a Ca2+-leak channel: cell biological and pathological consequences

Ca(2+) release via intracellular release channels, IP(3)Rs (inositol 1,4,5-trisphosphate receptors) and RyRs (ryanodine receptors), is perhaps the most ubiquitous and versatile cellular signalling mechanism, and is involved in a vast number of cellular processes. In addition to this classical release pathway there is limited, but yet persistent, information about less well-defined Ca(2+)-leak pathways that may play an important role in the control of the Ca(2+) load of the endo(sarco)plasmic reticulum. The mechanisms responsible for this 'basal' leak are not known, but recent data suggest that both IP(3)Rs and RyRs may also operate as Ca(2+)-leak channels, particularly in pathological conditions. Proteolytic cleavage or biochemical modification (such as hyperphosphorylation or nitrosylation), for example, occurring during conditions of cell stress or apoptosis, can functionally uncouple the cytoplasmic control domains from the channel domain of the receptor. Highly significant information has been obtained from studies of malfunctioning channels in various disorders; for example, RyRs in cardiac malfunction or genetic muscle diseases and IP(3)Rs in neurodegenerative diseases. In this review we aim to summarize the existing information about functionally uncoupled IP(3)R and RyR channels, and to discuss the concept that those channels can participate in Ca(2+)-leak pathways.     

Nonviral transfection of human umbilical cord blood dendritic cells is feasible, but the yield of dendritic cells with transgene expression limits the application of this method in cancer immunotherapy

Dendritic cells (DC) generated from human umbilical cord blood might replace patients' DC in attempts to elicit tumor-specific immune response in cancer patients. We studied the efficiency of transfection of human cord blood DC with plasmid DNA carrying the enhanced version of green fluorescent protein (EGFP) as a reporter gene, to test if nonviral gene transfer would be a method to load DC with protein antigens for immunotherapy purposes. Cord blood mononuclear cells were cultured in serum-free medium in the presence of granulocyte-monocyte colony stimulating factor (GM-CSF), stem cell factor (SCF) and Flt-3 ligand (FL), to generate DC from their precursors, and thereafter transfected by electroporation. Maturation of DC was induced by stimulation with GM-CSF, SCF, FL and phorbol myristate acetate (PMA). Transfected DC strongly expressed EGFP, but transfection efficiency of DC, defined as HLA-DR(+) cells lacking lineage-specific markers, did not exceed 2.5%. Expression of the reporter gene was also demonstrated in the DC generated from transfected, purified CD34(+) cord blood cells, by stimulation with GM-CSF, SCF, FL, and tumor necrosis factor alpha (TNF-alpha). Transfection of CD34(+) cells was very efficient, but proliferation of the transfected cells was much reduced as compared to the untransfected cells. Therefore, the yield of transgene-expressing DC was relatively low. In conclusion, nonviral transfection of cord blood DC proved feasible, but considering the requirements for immunotherapy in cancer patients, transfection of differentiated DC or generation of DC from transfected hematopoietic stem cells provide only a limited number of DC expressing the transgene.     

Wavelet analysis of scaling properties of gastric electrical activity.

We present a novel approach to the analysis of fluctuations in human myoelectrical gastric activity measured noninvasively from the surface of the abdomen. The time intervals between successive maxima of the wavelet transformed quasi-periodic electrogastrographic waveform define the gastric rate variability (GRV) time series. By using the method of average wavelet coefficients, the statistical fluctuations in the GRV signal in healthy individuals are determined to scale in time. Such scaling was previously found in a variety of physiological phenomena, all of which support the hypothesis that physiological dynamics utilize fractal time series. We determine the scaling index in a cohort of 17 healthy individuals to be 0.80 +/- 0.14, which compared with a set of surrogate data is found to be significant at the level P < 0.01. We also determined that the dynamical pattern, so evident in the spectrum of average wavelet coefficients of the GRV time series of healthy individuals, is significantly reduced in a cohort of systemic sclerosis patients having a scaling index 0.64 +/- 0.17. These results imply that the long-term memory in GRV time series is significantly reduced from healthy individuals to those with systemic sclerosis. Consequently, this disease degrades the complexity of the underlying gastrointestinal control system and this degradation is manifest in the loss of scaling in the GRV time series.     

Polycystin-2 Activation by Inositol 1,4,5-Trisphosphate-induced Ca2+ Release Requires Its Direct Association with the Inositol 1,4,5-Trisphosphate Receptor in a Signaling Microdomain

Autosomal dominant polycystic kidney disease is characterized by the loss-of-function of a signaling complex involving polycystin-1 and polycystin-2 (TRPP2, an ion channel of the TRP superfamily), resulting in a disturbance in intracellular Ca²⁺ signaling. Here, we identified the molecular determinants of the interaction between TRPP2 and the inositol 1,4,5-trisphosphate receptor (IP₃R), an intracellular Ca²⁺ channel in the endoplasmic reticulum. Glutathione S-transferase pulldown experiments combined with mutational analysis led to the identification of an acidic cluster in the C-terminal cytoplasmic tail of TRPP2 and a cluster of positively charged residues in the N-terminal ligand-binding domain of the IP₃R as directly responsible for the interaction. To investigate the functional relevance of TRPP2 in the endoplasmic reticulum, we re-introduced the protein in TRPP2^−/− mouse renal epithelial cells using an adenoviral expression system. The presence of TRPP2 resulted in an increased agonist-induced intracellular Ca²⁺ release in intact cells and IP₃-induced Ca²⁺ release in permeabilized cells. Using pathological mutants of TRPP2, R740X and D509V, and competing peptides, we demonstrated that TRPP2 amplified the Ca²⁺ signal by a local Ca²⁺-induced Ca²⁺-release mechanism, which only occurred in the presence of the TRPP2-IP₃R interaction, and not via altered IP₃R channel activity. Moreover, our results indicate that this interaction was instrumental in the formation of Ca²⁺ microdomains necessary for initiating Ca²⁺-induced Ca²⁺ release. The data strongly suggest that defects in this mechanism may account for the altered Ca²⁺ signaling associated with pathological TRPP2 mutations and therefore contribute to the development of autosomal dominant polycystic kidney disease.     

SPCA1 pumps and Hailey-Hailey disease

Both the endoplasmic reticulum and the Golgi apparatus are agonist-sensitive intracellular Ca2+ stores. The Golgi apparatus has Ca2+-release channels and a Ca2+-uptake mechanism consisting of sarco(endo)plasmic-reticulum Ca2+-ATPases (SERCA) and secretory-pathway Ca2+-ATPases (SPCA). SPCA1 has been shown to transport both Ca2+ and Mn2+ in the Golgi lumen and therefore plays an important role in the cytosolic and intra-Golgi Ca2+ and Mn2+ homeostasis. Human genetic studies have provided new information on the physiological role of SPCA1. Loss of one functional copy of the SPCA1 (ATP2C1) gene causes Hailey-Hailey disease, a skin disorder arising in the adult age with recurrent vesicles and erosions in the flexural areas. Here, we review recent experimental evidence showing that the Golgi apparatus plays a much more important role in intracellular ion homeostasis than previously anticipated.     

Similar Ca(2+)-signaling properties in keratinocytes and in COS-1 cells overexpressing the secretory-pathway Ca(2+)-ATPase SPCA1

Mutations in the ubiquitously expressed secretory-pathway Ca(2+)-ATPase (SPCA1) Ca(2+) pump result in Hailey-Hailey disease, which almost exclusively affects the epidermal part of the skin. We have studied Ca(2+) signaling in human keratinocytes by measuring the free Ca(2+) concentration in the cytoplasm and in the lumen of both the Golgi apparatus and the endoplasmic reticulum. These signals were compared with those recorded in SPCA1-overexpressing and control COS-1 cells. Both the sarco(endo)plasmic-reticulum Ca(2+)-ATPase (SERCA) and SPCA1 can mediate Ca(2+) uptake into the Golgi stacks. Our results indicate that keratinocytes mainly used the SPCA1 Ca(2+) pump to load the Golgi complex with Ca(2+) whereas the SERCA Ca(2+) pump was mainly used in control COS-1 cells. Cytosolic Ca(2+) signals in keratinocytes induced by extracellular ATP or capacitative Ca(2+) entry were characterized by an unusually long latency reflecting extra Ca(2+) buffering by an SPCA1-containing Ca(2+) store, similarly as in SPCA1-overexpressing COS-1 cells. Removal of extracellular Ca(2+) elicited spontaneous cytosolic Ca(2+) transients in keratinocytes, similarly as in SPCA1-overexpressing COS-1 cells. With respect to Ca(2+) signaling keratinocytes and SPCA1-overexpressing COS-1 cells therefore behaved similarly but differed from control COS-1 cells. The relatively large contribution of the SPCA1 pumps for loading the Golgi stores with Ca(2+) in keratinocytes may, at least partially, explain why mutations in the SPCA1 gene preferentially affect the skin in Hailey-Hailey patients.     

Phosphorylation of IP3R1 and the regulation of [Ca2+]i responses at fertilization: a role for the MAP kinase pathway.

A sperm-induced intracellular Ca2+ signal ([Ca2+]i) underlies the initiation of embryo development in most species studied to date. The inositol 1,4,5 trisphosphate receptor type 1 (IP3R1) in mammals, or its homologue in other species, is thought to mediate the majority of this Ca2+ release. IP3R1-mediated Ca2+ release is regulated during oocyte maturation such that it reaches maximal effectiveness at the time of fertilization, which, in mammalian eggs, occurs at the metaphase stage of the second meiosis (MII). Consistent with this, the [Ca2+]i oscillations associated with fertilization in these species occur most prominently during the MII stage. In this study, we have examined the molecular underpinnings of IP3R1 function in eggs. Using mouse and Xenopus eggs, we show that IP3R1 is phosphorylated during both maturation and the first cell cycle at a MPM2-detectable epitope(s), which is known to be a target of kinases controlling the cell cycle. In vitro phosphorylation studies reveal that MAPK/ERK2, one of the M-phase kinases, phosphorylates IP3R1 at at least one highly conserved site, and that its mutation abrogates IP3R1 phosphorylation in this domain. Our studies also found that activation of the MAPK/ERK pathway is required for the IP3R1 MPM2 reactivity observed in mouse eggs, and that eggs deprived of the MAPK/ERK pathway during maturation fail to mount normal [Ca2+]i oscillations in response to agonists and show compromised IP3R1 function. These findings identify IP3R1 phosphorylation by M-phase kinases as a regulatory mechanism of IP3R1 function in eggs that serves to optimize [Ca2+]i release at fertilization.     

Compositional dynamics of natural forests in the Bialowieza National Park,northeastern Poland

Abstract. This paper presents results of a long-term study on natural forest dynamics in the Bia?owieza National Park (BNP), northeastern Poland. Five permanent sample areas were used, each consisting of a transect of varying width (40 - 60 m) and length (200 - 1380 m). The total sample area is 14.9 ha. The study covers the period 1936–1992. During this period measurements were made on five occasions at approximately 10-yr intervals. On each measurement date all trees with DBH > 5 cm were identified and their spatial location, diameter, crown condition and position in the canopy determined. During the study period the stands underwent noticeable changes, mainly in terms of tree species composition. The major change was a quantitative increase of the late-successional species: Tilia cordata and Carpinus betulus, also to a lesser degree Fraxinus excelsior and, in the last period, of the early successional Alnus glutinosa. Declining species included both early- and late-succession species. Among the latter group, Picea abies ranked first. This species lost much of its importance during the last few decades. P. abies was followed by Pinus sylvestris which is an important component of the climax vegetation under the conditions prevailing in Bialowieza, at least on more oligotrophic sites. Still, this species has not been able to regenerate during the whole study period. Some other late-succession species, Acer platanoides and Quercus robur, were also amongst the declining species. Although the basal area of Q. robur increased, its population was getting older and the process of natural regeneration was markedly impeded. All typical pioneer, short-lived species: Betula pendula and B. pubescens, Salix caprea and Populus tremula also decreased, which was probably caused by a lack of major disturbances during the study period. In general, the results obtained for the semi-natural conditions of Bialowieza during the 56-yr observation period suggest a rather high compositional instability of the forest stands there. A more precise identification of the role of particular factors in the observed stand dynamics is difficult because of the paucity of appropriate historical and environmental data which refer directly to the study plots; moreover, the data are generally incompatible and of different resolution.     

Influence of 50 Hz magnetic field on human heart rate variability: linear and nonlinear analysis

This study investigated the problem of the influence of 50 Hz magnetic field (MF) on human heart rate variability (HRV). The exposure system was a commercial device for magnetotherapy, generating field of the strength of 500 microT at the center of the coil, 150-200 microT at the position of human subjects' heart and 20-30 microT at the position of subjects' head. The exposure protocols, applied randomly, were either "half hour MF-off/half hour MF-on" or "half hour MF-off/half hour MF-off." The phonocardiographic (PhCG) signal of 15 volunteers were obtained during exposure and used for calculation of time-domain HRV parameters (mean time between heart beats (N-N), standard deviation of time between heart beats (SDNN), and the number of differences of successive beat-to-beat intervals greater than 50 ms, divided by the total number of beat-to-beat intervals (pNN50)) and nonlinear HRV measures (approximate entropy (ApEn), detrended fluctuation scaling exponents). The protocol MF-off/MF-on was applied in nine subjects. Repeated measures ANOVA (RMANOVA) performed for Mf-off/MF-off protocol indicated no statistical difference among four 15 min intervals of HRV data (P value >20% for all parameters except for N-N, where P = 3.7%). RMANOVA followed by the post hoc Tukey test performed for Mf-off/MF-on protocol indicated a statistically significant difference during MF on for N-N (8% increase, P <.1%), SDNN (40% increase, P = 1.1%), and pNN50 (110% increase, P <.1%). The results of the analysis indicate that the changes of these parameters could be associated with the influence of MF.